This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Candida albicans is a prevalent human pathogen, which presents a serious health risk in immunocompromised individuals. The first line of host defense against C. albicans involves innate immune responses that challenge the invading pathogen with macrophage- and neutrophil-generated nitrosative and oxidative stress. The counter-measures that pathogens muster against this stress are not well understood, but appear to include roles for proteins involved in enzymatic detoxification of stress-inducing chemical agents, and in signaling cascades that regulate of cell growth. Very little is known, however, at the molecular level, of the responses of C. albicans to oxidative and nitrosative stress. The broad goal of this research is to develop enhanced differential proteomics methods and to apply them to advance our understanding of the molecular bases of C. albicans defenses against host nitrosative and oxidative challenges. Elucidation of the primary biochemical steps in defense pathways will establish new targets for the design of more effective and specific drugs against C. albicans. This research will focus on early responses that are likely to involve reversible modification of reactive protein thiols that yield S-nitrosothiols (RSNO), S- sulfenic acids (RSOH) and transient disulfices. The fundamental questions that we will address are: (1) what components of the C. albicans proteome are modified by different types and levels of oxidative and nitrosative stress;(2) which of these modifications involve redox signaling processes in which protein sensors detect and transduce the stress;and (3) what biochemical pathways are modulated by stress-responsive protein modifications? Differential thiol redox profiling (dThiRP) - featuring a new generation of "Zdyes", that promise unsurpassed threshold sensitivities and the capacity for multi-color differential profiling - will be used to address these questions. Specifically, the research program entails: (1) validation of thiol "switch" detection chemistry with the Zdye labels;(2) global proteomic measurements of changes in protein levels, S-nitrosothiols, sulfenic acids, and disulfides in C. albicans cells subject to exogenous chemical, and macrophage challenge;(3) measurements of the relative resistance to chemical and macrophage challenge of homozygous and heterozygous KO strains, suggested by modeling of the wild-type proteomic data.